Abstract

The development of potent steroid sulfatase inhibitors is an important
new therapeutic strategy for the treatment of postmenopausal women with
breast cancer. A series of tricyclic coumarin sulfamates were
synthesized, and their inhibitory properties were examined in
vitro and in vivo. In a placental microsomal
assay system, 667 COUMATE emerged as the most potent inhibitor with an
IC50 of 8 nm. Administration of a single dose
(10 mg/kg, p.o.) of 667 COUMATE inhibited rat liver estrone sulfatase
activity by 93%. 667 COUMATE was devoid of estrogenicity, as indicated
by its failure to stimulate the growth of uteri in ovariectomized rats.
In vivo, estrone sulfate-stimulated growth of uteri in
ovariectomized rats was inhibited by 667 COUMATE. Using the
nitrosomethylurea-induced mammary tumor model, we found that 667
COUMATE caused regression of estrone sulfate-stimulated tumor growth in
a dose-dependent manner. The identification of 667 COUMATE as a potent
steroid sulfatase inhibitor will enable the therapeutic potential of
this type of therapy to be evaluated.

Introduction

The development of enzyme inhibitors to block estrogen synthesis
offers a new approach for the treatment of postmenopausal women with
hormone-dependent breast tumors. Thus far, research has been directed
mainly toward the identification of aromatase inhibitors, which block
the conversion of androstenedione to estrone
(1, 2)
. There
is, however, a growing awareness of the role that steroid sulfatase,
which hydrolyses
E1S
3
to estrone, may have in regulating the formation of estrogenic steroids
(3,
4,
5)
. The steroid sulfatase that hydrolyses estrone
sulfate is also responsible for the formation of DHEA from DHEA sulfate
(6)
. Reduction of DHEA gives rise to Adiol which, although
an androgen, can bind to the estrogen receptor. Adiol can stimulate the
growth of breast cancer cells in vitro and
carcinogen-induced mammary tumors in vivo in ovariectomized
rats
(7, 8)
. The E1S surrogate, EMATE, was identified as a
potent, active site-directed irreversible inhibitor
(9, 10)
. EMATE was active in vivo and had a prolonged
duration of action
(11)
but, unexpectedly, EMATE proved to
be a potent estrogen, being five times more active than
ethinylestradiol on oral application to rats
(12)
. A
series of structure-activity relationship studies revealed that the
active pharmacophore required for potent steroid sulfatase inhibition
is a sulfamate group attached to an aromatic ring
(13)
.
This finding led to the design and synthesis of a number of sulfamated
1-4 ring compounds that are potent non-estrogenic steroid sulfatase
inhibitors
(14,
15,
16)
. Such inhibitors included COUMATE
which, although less potent than EMATE, was active in vivo(15)
. In this paper, we report on the in vitro
and in vivo activity of a number of tricyclic coumarin
sulfamates, one of which was selected for testing in an induced mammary
tumor model in rats.

Materials and Methods

Synthesis of Tricyclic Coumarin and Oxepin Sulfamates.

The series of 665–668 tricyclic coumarins were prepared by a
Pechmann synthesis of the starting coumarin by reacting resorcinol
with the corresponding 5–8-membered cyclic β-ketoester
(e.g., methyl 2-oxocycloheptane carboxylate for 667
COUMARIN) in the presence of concentrated sulfuric and trifluoroacetic
acids, The tricyclic oxepin was prepared in a similar manner from
resorcinol and ethyl (2-oxocyclohexyl) acetate.

In Vitro and in Vivo Inhibition of
Estrone Sulfatase Activity.

The ability of the tricyclic coumarin and oxepin sulfamates to inhibit
E1-STS activity and determinations of IC50s were
carried out using placental microsomes (100,000 × g fraction; Ref.
17
). For in vivo
studies, female Wistar rats (Harlan Olac, Bicester, Oxon, United
Kingdom) were treated p.o. with vehicle (propylene glycol) or drug
(0.1–1.0 mg/kg) with either a single dose or five multiple doses at
daily intervals.

Uterotrophic Study.

To examine in vivo for possible estrogenic effects of 667
COUMATE, rats were ovariectomized and 14 days later received vehicle
(propylene glycol, 200 μl, p.o.) or 667 COUMATE (2 mg/kg/day, p.o.)
for 5 days. The ability of 667 COUMATE to inhibit E1S-stimulated
uterine growth in ovariectomized animals was also investigated. For
this, animals received either vehicle or 667 COUMATE (2 mg/kg/day,
p.o.) initially for 2 days to suppress E1-STS activity. Animals either
continued to receive vehicle p.o. plus E1S (50 μg/day, s.c.) or 667
COUMATE (2 mg/kg/day, p.o.) plus E1S (50 μg/day, s.c.) for another 5
days.

Animals were killed 24 h after administration of the last dose of
drug, and uteri were excised of fat and weighed. Total body weights of
the animals were also recorded, and the results were expressed as
uterine weight × 100/total body weight.

Inhibition of Mammary Tumor Growth.

Ludwig rats were obtained from Harlan Olac after induction of mammary
tumors with NMU
(16)
. Tumor development was monitored, and
animals were ovariectomized when tumors reached 0.8–1.5 cm in
diameter. Tumors were allowed to regress over a 12- to 13-day period to
confirm their hormone-dependent status. Regrowth of tumors was
stimulated with E1S (50 μg/day, s.c.). When tumors had regrown,
animals continued to receive either E1S alone or E1S plus 667 COUMATE
at 10 mg/kg/day or 2 mg/kg/day, p.o., until tumor regression had
occurred. Tumor volumes were calculated from two measured diameters
(11)
.

Estrone Sulfatase Activity in Tissues.

Liver and tumor tissues obtained from rats were immediately frozen on
solid carbon dioxide and stored at −20°C until assayed. Tissues were
homogenized and, after centrifugation to remove cell debris, aliquots
of the supernatant were used for the sulfatase assay
(17)
.

Statistics.

Student’s t test was used to assess the significance of the
effect of 667 COUMATE on uterine growth. The paired Student’s
t test was used to assess the significance of changes in
tumor volumes after ovariectomy, E1S-stimulated regrowth, and after
inhibitor treatment.

Results and Discussion

Inhibition of E1-STS Activity in Vitro and in
Vivo.

The series of tricyclic coumarin and oxepin sulfamates were all
potent inhibitors of in vitro E1-STS activity (Fig. 2)
⇓
, with almost complete (91–99%) inhibition being achieved at 1μ
m. Determination of
IC50s for this series revealed that 667 COUMATE
was the most potent, with an IC50 of 8
nm. IC50s for the other
derivatives were 200, 70, and 30 nm for the 665,
666, and 668 COUMATES and 250 nm for the 676
OXEPIN sulfamate. The relative potency of 667 COUMATE to that of
COUMATE and EMATE was assessed using the placental microsome assay.
The IC50 for EMATE was 25
nm and for COUMATE, 800 nm.
Thus, 667 COUMATE (IC50, 8
nm) is three times more potent than EMATE and 100
times more potent than COUMATE in inhibiting E1-STS activity.

Inactivation of E1-STS activity in placental microsomes by
665 COUMATE, 666 COUMATE, 667 COUMATE, 668 COUMATE, and 676 OXEPIN.[
3H]E1S adjusted to 20 μm with unlabeled
substrate with or without inhibitors at concentrations of 0.01–1.0μ
m was incubated with placental microsomes (125 μg of
protein/ml) for 30 min. The product formed was isolated by toluene
extraction with [4-14C]estrone being used to monitor
procedural losses. Each point represents the mean of
triplicate measurements for which the coefficients of variation were<10%.

On the basis of its highest in vitro inhibitory
potency, 667 COUMATE was selected for testing in vivo.
Single or multiple doses of 667 COUMATE (1 mg/kg) inhibited rat liver
E1-STS activity by >90% (data not shown). 667 COUMATE, therefore, has
a similar potency in vivo to that of EMATE
(11)
. The duration of inhibition of E1-STS activity by 667
COUMATE was assessed using samples of liver tissue obtained 1, 3, and 7
days after a single dose (10 mg/kg). Complete recovery of E1-STS
activity had occurred by 7 days after drug administration (data not
shown). The in vitro and in vivo hydrolysis of
DHEA-sulfate was also efficiently inhibited by 667 COUMATE (data not
shown). 667 COUMATE has been identified as a potent nonsteroidal
inhibitor of steroid sulfatase and is considerably more potent then the
two-ringed COUMATE. The two rings of COUMATE were thought to act as
mimics for the A and B rings of the steroid nucleus. The introduction
of a third ring in the tricyclic coumarin sulfamates has increased the
potency of 667 COUMATE 100-fold compared with that of COUMATE. EMATE
and COUMATE have been shown previously to act in a time- and
concentration-dependent manner
(10, 13)
. 667 COUMATE also
inhibits the enzyme in a similar manner, but the inactivation of E1-STS
by this inhibitor was more rapid than by EMATE (data not shown),
confirming its enhanced potency. Although the mechanism by which 667
COUMATE inactivates E1-STS is not yet fully elucidated, it has been
postulated that, like EMATE, it acts via irreversible sulfamoylation of
one or more residues in the enzyme active site.

Although 667 COUMATE has a similar potency to that of EMATE in
vivo, there is a marked difference in the time taken for E1-STS
activity to be restored after a single dose of these drugs. For 667
COUMATE, similar to COUMATE
(15)
, E1-STS activity had
recovered by 7 days. This is much shorter than the recovery period seen
after EMATE administration, which only increased by 10–15% 15 days
after cessation of the drug
(11)
.

Inhibition of E1S-stimulated Uterine and Tumor Growth.

Although previous studies revealed that 667 COUMATE did not stimulate
the growth of estrogen-sensitive MCF-7 breast cells in vitro(18)
, it was tested for estrogenicity in vivo
using the rat uterotrophic model. In ovariectomized rats receiving E1S
(50 μg/day, s.c.) for 5 days, uterine growth was stimulated by 170%
compared with animals receiving vehicle (Fig. 3)
⇓
. In contrast, for animals receiving 667 COUMATE (2 mg/kg, p.o.) for 5
days, uterine weights did not differ significantly from those of
control animals. This model was also used to test the ability of 667
COUMATE to inhibit E1S-stimulated uterine growth. Measurements of
uterine weights 24 h after administration of the last dose of drug
and E1S revealed that the ability of E1S to stimulate uterine growth
was blocked by the co-administration of 667 COUMATE (Fig. 3)
⇓
. Thus,
although 667 COUMATE is a potent inhibitor of E1-STS activity, unlike
EMATE it is devoid of estrogenicity. It was evident, however, from this
model system that 667 COUMATE was effective in blocking the ability of
E1S to stimulate uterine growth.

Since the identification of EMATE as a steroid sulfatase inhibitor,
there has been considerable progress in developing potent,
non-estrogenic inhibitors. Such compounds include modifications at the
C17 position of EMATE to give a series of alkylamido and
N-alkylcarbamoyl sulfamates
(19)
or
17α-p-tert-butylbenzyl sulfamates
(20)
. The development of 667 COUMATE represents an
important advance in identifying a nonsteroidal inhibitor that,
although three times more potent than EMATE in vitro, is not
estrogenic. 667 COUMATE has been identified as a potent inhibitor with
therapeutic potential and is scheduled to enter a Phase I clinical
trial for use in postmenopausal women with breast cancer in the near
future.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵2 To whom requests for reprints should be
addressed, at Endocrinology and Metabolic Medicine, Imperial College
School of Medicine, St. Mary’s Hospital, London W2 1NY, United
Kingdom. Telephone: 44-207-886-1738; Fax: 44-207-886-1790; E-mail: m.reed{at}ic.ac.uk